CN114480354A

CN114480354A - Method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by utilizing genetically engineered rice

Info

Publication number: CN114480354A
Application number: CN202011156258.2A
Authority: CN
Inventors: 杨代常; 董亮亮; 李坤鹏; 尹恒
Original assignee: Wuhan Healthgen Biotechnology Co Ltd
Current assignee: Wuhan Healthgen Biotechnology Co Ltd
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2022-05-13
Also published as: WO2022089340A1

Abstract

The invention discloses a preparation method of genetically engineered rice for efficiently expressing human chymotrypsinogen and a method for preparing recombinant human chymotrypsin from the genetically engineered rice for expressing the human chymotrypsinogen. Processing and crushing genetically engineered rice which efficiently expresses a human chymotrypsinogen gene optimized by a rice codon, mixing the crushed genetically engineered rice with an extraction buffer solution, and extracting and filtering to obtain a crude extract containing a recombinant human chymotrypsinogen; and (3) subjecting the crude extract containing the recombinant human chymotrypsinogen to cation exchange chromatography and anion exchange chromatography to obtain a recombinant human chymotrypsinogen purification target substance. The recombinant human chymotrypsinogen is activated by trypsin, and the recombinant human chymotrypsin is obtained after freeze drying. The method has the advantages of simple operation, no animal-derived material, high purity and activity of the obtained chymotrypsin, low cost, and easy industrial amplification.

Description

Method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by utilizing genetically engineered rice

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by taking genetically engineered rice as a bioreactor.

Background

Chymotrypsin (Chymotrypsin), also called Chymotrypsin, belongs to the family of serine proteases and specifically hydrolyzes peptide bonds formed by aromatic amino acid carboxyl groups. The chymotrypsin is secreted by pancreas, enters small intestine along with pancreatic juice, is cut into two parts connected by disulfide bond between Arg15 and Ile16 (numbered according to the sequence of Bovine chytrysin A) by trypsin, then the chymotrypsin cuts off short peptide Ser14-Arg15 at Leu13 and cuts off short peptide Tyr147-Asn148 at Tyr146 and Asn148, and three polypeptide chains connected by disulfide bond are formed and have biological activity.

The chymotrypsin has wide clinical application, has pharmacological action of decomposing protein, promoting the removal of blood clots, purulent secretion, necrotic tissues and the like, and is mainly used for relaxing ciliary ligaments and relieving traumatic iridocyclitis in ophthalmic surgery; it can also be used for wound or local inflammation to reduce local secretion and edema.

Because chymotrypsin can digest various proteins, the chymotrypsin can degrade proteins which maintain the life of host cells when expressed by other expression systems, and the expression level of the existing expression system is not high. Therefore, the existing chymotrypsin preparation is still extracted from the pancreas of cattle or pigs, is prepared into chymotrypsinogen after fractional salting-out, is activated by trypsin and is refined by ammonium sulfate and ethanol recrystallization. The process has the advantages of poor extraction specificity, long operation time and low yield. The chymotrypsin extracted from the pancreas of cattle or pigs has an amino acid sequence homology of 82% with the amino acid sequence of human chymotrypsin and bovine chymotrypsin and an amino acid sequence homology of only 43% with the amino acid sequence of porcine chymotrypsin. The existing animal source chymotrypsin has the risk of introducing animal source viruses, and can cause immune reaction after heterologous protein enters a human body, so that the safety risk of medication is greatly increased.

Human chymotrypsinogen is a peptide chain containing 263 amino acids, reaches the extracellular part through a signal peptide during the in vivo activation process, and the number of amino acids contained in a protein molecule is changed into 245 after the signal peptide is hydrolyzed by peptidase, namely the chymotrypsinogen. Chymotrypsinogen is not activated to the active form by trypsin until it is transported to the small intestine.

The development trend of the biological medicine industry is that no biological products of animal origin and humanization exist. The first non-animal-derived recombinant human insulin is extracted from animal pancreas in 1982, the first non-plasma product recombinant thrombin is marketed in 2008, and the first non-animal-derived plant-derived recombinant human serum albumin is subjected to phase 1 clinical research in the United states in 2020. The development of animal origin-free and humanized chymotrypsin conforms to the development trend of biological medicines.

At present, no recombinant human chymotrypsin pharmaceutical product is on the market at home and abroad. The patent CN104342423A discloses a preparation method and application of high-activity recombinant human chymotrypsin, which adopts escherichia coli to express an inclusion body of the recombinant human chymotrypsin, and obtains the recombinant human chymotrypsin with higher purity through ion exchange chromatography after denaturation and renaturation treatment.

Disclosure of Invention

The invention utilizes a rice endosperm cell bioreactor to efficiently express recombinant human chymotrypsinogen, extracts and purifies the recombinant human chymotrypsinogen from genetically engineered rice, and then obtains the recombinant human chymotrypsinogen with bioactivity by activation.

One purpose of the invention is to provide a method for efficiently expressing recombinant human chymotrypsinogen in genetically engineered rice, which comprises the following steps:

(1) synthesizing a rice codon optimized human chymotrypsin pro-gene sequence shown as SEQ ID NO. 1;

(2) constructing a recombinant human chymotrypsinogen expression vector for rice endosperm cell specific expression;

(3) transforming the vector obtained in the step 2 into callus regeneration tissues of rice varieties;

(4) culturing the callus regeneration tissue, and obtaining a transgenic recombinant human chymotrypsin proto-gene engineering rice plant through screening and induction;

(5) culturing the recombinant human chymotrypsin proto-gene engineering rice plant to obtain the recombinant human chymotrypsin proto-gene engineering rice.

Wherein the recombinant human chymotrypsinogen expression vector preferably has a structure as shown in figure 3.

Another object of the present invention is to provide a method for preparing recombinant human chymotrypsin from genetically engineered rice of recombinant human chymotrypsin pro-gene, comprising the steps of:

the method for extracting and purifying the recombinant human chymotrypsinogen comprises the following steps:

(1) extracting a crude extract containing recombinant human chymotrypsinogen from recombinant human chymotrypsinogen genetically engineered rice;

(2) carrying out Unigel80 SP cation exchange chromatography on the crude extract containing the recombinant human chymotrypsinogen to obtain a primary product;

(3) subjecting the primary product to Unigel80Q anion exchange chromatography to obtain purified recombinant human chymotrypsinogen;

and (3) activating the purified recombinant human chymotrypsinogen by trypsin, and freeze-drying to obtain the recombinant human chymotrypsin.

Further, the method for extracting and purifying the recombinant human chymotrypsinogen comprises the following steps:

1) the recombinant human chymotrypsin pro-genetic engineering paddy is hulled and processed into semi-polished rice, and ground into rice flour of 80-100 meshes. Rice flour was mixed with extraction buffer at a ratio of 1:5 (weight/volume, kg/L), extracting at 4-30 ℃ for 1-16 h, adding 2-5% perlite for filter pressing, and filtering the filtrate with a 0.22 mu m filter membrane to obtain the crude extract of the recombinant human chymotrypsinogen. The extraction buffer solution comprises the following components: 4.44-69.66 g/L disodium hydrogen phosphate dodecahydrate, 0.58-19.71 g/L citric acid monohydrate, and pH 2.5-8.0.

2) The primary separation and purification is carried out using cationic chromatographic media including SP Bestrose FF (Bogelong (Shanghai) Biotechnology Co., Ltd.), Unigel80 SP (Suzhou nahcon MicroTechnique Co., Ltd.), Unigel80CM (Suzhou nahcon MicroTechnique Co., Ltd.), and Unigel MMC50S (Suzhou nahcon MicroTechnique Co., Ltd.). Preferably, Unigel80 SP chromatography media is used. In one embodiment, the chromatography column is equilibrated at a linear flow rate of 300-600 cm/h using 4-6 Column Volumes (CV) of a disodium hydrogen phosphate-citric acid buffer having a pH of 3.0-3.5; taking the crude extract obtained in the step 1) as a loading solution, wherein the pH of the loading solution is 3.0-3.5, and the loading volume is not more than 74 CV; eluting the impurity protein by using a disodium hydrogen phosphate-citric acid buffer solution with the column volume of 5-10 times and the pH value of 3.5-4.0 at a linear flow rate of 300-600 cm/h; eluting the recombinant human chymotrypsinogen by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 3.9-4.1 and containing 100-200 mM sodium chloride at a linear flow rate of 300-600 cm/h, and collecting an eluent containing the recombinant human chymotrypsinogen to obtain a primary product containing the recombinant human chymotrypsinogen;

3) the precision purification was performed using anionic chromatography media including Unigel80Q (Suzhou nano micro technology, Inc.), Nanogel 50Q (Suzhou nano micro technology, Inc.), MMA-50S (Suzhou nano micro technology, Inc.), Q Bestrose FF (Bogelong (Shanghai) Biotechnology, Inc.), Unigel 30Q (Suzhou nano micro technology, Inc.), Q Bestrose HP (Bogelong (Shanghai) Biotechnology, Inc.). Preferably, Unigel80Q chromatography media is used. The method comprises the following steps of (1) balancing a column at a flow rate of 382-840 cm/h by using a disodium hydrogen phosphate-citric acid buffer solution which is 10-15 times of the column volume, has a pH of 7.9-8.1 and has a conductivity of 1.5-2.0 mS/cm; properly concentrating the eluent containing the recombinant human chymotrypsinogen in the step 2) by using a 5-10 kDa ultrafiltration membrane (polyether sulfone material), dialyzing by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 7.9-8.1 and the conductance of 1.5-2.0 mS/cm until the conductance is reduced to 1.5-2.5 mS/cm, and adjusting the pH to 7.9-8.1 to serve as a sample loading solution for the chromatography; eluting with a disodium hydrogen phosphate-citric acid buffer solution containing 25-75 mM sodium chloride at the flow rate of 382-840 cm/h and with the pH of 6.5-8.0, and collecting an eluent containing the recombinant human chymotrypsinogen to obtain the recombinant human chymotrypsinogen with the purity of more than 95%.

Further, the method for activating the recombinant human chymotrypsinogen provided by the application comprises the following steps:

(1) adding an activating agent trypsin into 0.05-0.5% (w/w) of the recombinant human chymotrypsinogen solution of 0.75-4 mg/ml, standing and activating for 36-72 hours at the temperature of 2-8 ℃.

(2) And after the activation is finished, adjusting the pH value to 2.5-3.5 to stop the activation.

(3) And (3) properly concentrating the recombinant human chymotrypsin in the step (2) by using a 5-10 kDa ultrafiltration membrane (made of polyether sulfone material), dialyzing by using ultrapure water until the conductance is reduced to be less than 0.5mS/cm, and adjusting the pH to be 5.5-6.5. And (5) loading into a tray and freeze-drying.

The invention also provides a recombinant human chymotrypsinogen prepared by rice endosperm cell expression according to the method.

Compared with the prior art, the invention has the advantages and positive effects that:

the present inventor relies on two dominant platforms-rice endosperm cell protein expression platform (Oryz)^HiExp) And a protein purification platform (Oryz)^Pur) The rice endosperm cell is adopted to express the recombinant human chymotrypsinogen with high efficiency, and the recombinant human chymotrypsinogen with the SEC-HPLC purity higher than 99 percent can be obtained after extraction and two-step chromatography. The activity of the recombinant human chymotrypsin can reach more than 1500U/mg and is higher than the standard of 1000U/mg in 2020 edition Chinese pharmacopoeia. Compared with the traditional production process, the production process disclosed by the invention has the advantages of no animal-derived material, simplicity in operation, short production time, higher safety, environmental friendliness and important application prospect, and accords with the development trend of future biopharmaceuticals.

Drawings

FIG. 1 shows the plasmid structure of pOsPMP 799.

FIG. 2 shows the plasmid structure of pOsPMP 800.

FIG. 3 shows the plasmid structure of pOsPMP 801.

FIG. 4 shows PCR detection of target gene of genetically engineered rice plant. Wherein M is a DNA standard molecular weight Marker; 1-23 are different plants of T1 generation genetic engineering material; p is a positive control plasmid; the red arrow indicates the gene of interest.

FIG. 5 shows the detection of rice expression level of some strains of genetically engineered rice. Wherein M is a DNA standard molecular weight Marker; 2. 7, 8, 9, 16 and 20 respectively represent different strains in the process of breeding the genetically engineered rice; the red arrow indicates the target protein.

FIG. 6 shows SDS-PAGE detection results of crude recombinant human chymotrypsin extracts under different extraction conditions. Wherein M is a standard molecular weight Marker; 1 to 27 in A to C represent different extraction conditions of 1 to 27 in Table 1, respectively.

FIG. 7 shows the results of electrophoresis detection of crude recombinant human chymotrypsin extract at different pH, temperature and extraction time. Wherein M is a standard molecular weight Marker.

FIG. 8 SDS-PAGE of eluted fractions from Unigel80 SP, Unigel80CM and Unigel MMC50S chromatography. Wherein A is the SDS-PAGE detection result of the Unigel80 SP chromatographic elution component; b is the SDS-PAGE detection result of the Unigel80CM chromatographic elution component; c is SDS-PAGE detection result of Unigel 50S chromatographic elution components; m is a standard molecular weight Marker; wash denotes Wash; CIP1 and CIP2 denote regeneration 1 and regeneration 2, respectively; FT2 is the penetration of the trailing part at rebalancing.

FIG. 9 shows the results of Unigel80 SP penetrant liquid electrophoresis, where M is a standard molecular weight Marker, L is a chromatographic sample loading solution, and 25-36 represent the numbers of the collection tubes, respectively.

FIG. 10 SDS-PAGE of different salt concentrations of the eluate fractions at pH 4.0. Wherein M is a standard molecular weight Marker; w1-1 and W1-2 are used for collecting impurity washing peaks in a segmented manner; elu is elution and collection liquid; CIP as regenerating liquid

FIG. 11 shows the results of different anion chromatographic electrophoretic tests. Wherein FT is the transudate; 25. 50, 75, 100 and 150 respectively represent different elution components containing mM sodium chloride; the red arrow indicates the protein of interest.

FIG. 12 shows the results of SDS-PAGE detection of Unigel80Q chromatography fractions. Wherein M is a standard molecular weight marker; l is a sample loading solution; FT is transudate fluid; w is impurity washing collecting liquid; elu is elution and collection liquid; CIP is regeneration liquid; reducing, namely adding beta-mercaptoethanol in the electrophoresis sample preparation process, and non-reducing, namely not adding beta-mercaptoethanol in the sample preparation process.

FIG. 13 shows electrophoresis detection of Unigel80Q chromatography collection liquid under different elution conditions. Wherein 1 represents a 50mM sodium chloride elution pool at pH6.5 in a reduced state; 2 represents the 50mM NaCl eluate at pH6.5 under non-reducing conditions; 3 represents the pH7.0 in the reduced state, 50mM sodium chloride eluted pool; 4 represents the pH7.0 in the reduced state, 25mM sodium chloride eluted pool; 5 denotes pH 7.5 in the reduced state, 50mM sodium chloride elution pool; 6 denotes the pH8.0 in the reduced state, 50mM sodium chloride eluate.

FIG. 14. three lots of pilot process chromatograms. Wherein A is Unigel80 SP chromatogram and B is Unigel80Q chromatogram; 20200804, 20200805, 20200806 respectively represent different production lot numbers.

FIG. 15 shows the results of three small-scale electrophoretic tests (reduction), where M is a standard molecular weight Marker; batch 1 represents 20200804, batch 2 table 20200805, and batch 3 represents 20200806; reduction means that a reducing agent beta-mercaptoethanol is added in the sample preparation process.

FIG. 16 detection profile of recombinant human chymotrypsin SEC-HPLC before activation.

FIG. 17 detection profile of activated recombinant human chymotrypsin SEC-HPLC.

FIG. 18 SDS-PAGE detection of recombinant human chymotrypsin.

Detailed Description

The technical solutions of the present invention will be described in detail below by way of examples and figures to better illustrate the features and advantages of the present invention. The examples provided should be construed as illustrative of the method of the invention and not limiting the technical solutions disclosed in the invention in any way.

The reagents and instruments used in the following examples are all generally commercially available unless otherwise specified.

[ example 1 ] preparation of genetically engineered Rice highly expressing recombinant human chymotrypsinogen

1. Construction of recombinant human chymotrypsin protogene expression vector

In the embodiment, a rice specific promoter Gt13a and a signal peptide thereof are selected to mediate the expression of the recombinant human chymotrypsinogen gene in rice endosperm cells. According to the sequence of human chymotrypsin gene (Genbank accession number: NP-001897.4), Nanjing Kingsley Biotech limited was entrusted with the synthesis according to the preferred genetic codons of rice, specifically as shown in SEQ ID NO.1, the nucleotide was changed by 15% and the codon was changed by 35% after the codon optimization by the rice preferred codon, but the corresponding amino acid sequence was not changed, and the constructed plasmid was pOsPMP799 (FIG. 1). In the embodiment, a rice specific promoter Gt13a and a signal peptide thereof are selected to mediate the expression of the human chymotrypsinogen gene in rice endosperm cells. The synthesized codon optimized human chymotrypsin protogene (SEQ ID NO.1) is cut by MlyI and XhoI and then cloned into NaeI and XhoI cut pOsPMP003, and an intermediate vector plasmid pOsPMP800 (figure 2) is constructed by T4 ligase; then, pOsPMP800 was digested with HindIII and EcoRI, and the expression cassette containing the Gt13a promoter/signal peptide sequence, codon-optimized human chymotrypsin origin gene and Nos terminator, which was 2263bp in length, was inserted into HindIII and EcoRI digested binary expression vector pc1300 to construct an Agrobacterium-mediated bacterial plasmid, which was designated pOsPMP801 (FIG. 3).

2. Genetic engineering of rice

The pOsPMP801 plasmid was transformed into Agrobacterium tumefaciens EHA105 (Invitrogen, USA), pOsPMP801 was transformed into callus regeneration tissue of rice variety TP309 by Agrobacterium tumefaciens mediated transformation, and a complete plant was formed after cultivation, selection and induction. The specific method comprises the following steps:

(1) callus induction

1) Husking mature rice seeds, soaking in 70% alcohol, sterilizing for 1min, and treating with 20% sodium hypochlorite for 30 min;

2) washing with sterilized sterile water for 5-7 times;

3) inoculating the treated paddy rice to an induction culture medium (N6 culture medium), and inoculating 6-8 grains per dish;

4) the treatment was carried out at 32 ℃ for about 5 to 7 days.

(2) Preparation of Agrobacterium

The agrobacterium containing the expression vector pOsPMP801 is subjected to amplification culture. The strain is smeared on a kanamycin-resistant plate and cultured in an incubator at 28 ℃ for 2 to 3 days.

(3) A single colony of Agrobacterium was picked up by an inoculating loop and cultured in a suspension medium (AAM liquid medium) with shaking (160rpm) at 28 ℃. Typically 100ml of medium can be scraped into 3 to 4 loops using a inoculating loop.

(4) Agrobacterium infection (Co-culture)

1) Transferring the callus into a sterilized Erlenmeyer flask;

2) adjusting the OD600 value of the agrobacterium tumefaciens suspension to be between 0.05 and 0.1;

3) suspending the rice in AAM culture medium, infecting for 1.5min, and continuously shaking;

4) discarding the bacterial liquid, sucking the redundant bacterial liquid by using sterile filter paper, taking out the callus, placing the callus on the sterile filter paper, and draining for 30-45 min;

5) sterile filter paper was placed on 2N6-AS medium. Then 500. mu.l of AAM containing AS (acetosyringone, 250mg/ml) was dropped on sterile filter paper with a diameter of 9cm, and the infected calli were placed on the filter paper and cultured in the dark at 25 ℃ for 3 days.

(5) Washing and screening

1) Transferring the co-cultured callus into a sterilized Erlenmeyer flask;

2) washing the callus with sterilized water for 5-7 times;

3) soaking the infected callus in sterile water containing 0.5g/L of cefadriamycin for about 30min, and then shaking at the temperature of 28 ℃ and the speed of 200rpm for 20-30 min;

4) pouring out the sterilized water containing the antibiotics, and pouring the triangular flask into a sterilized culture dish containing filter paper for about 15 min;

5) drying the callus on sterilized filter paper;

6) transferring the callus to a selection medium containing HPT antibiotic and culturing for 20-30 days.

(6) Callus differentiation

After 20-30 days of selection, the callus with HPT resistance is transferred to a differentiation medium (N6 medium), and is cultured for 20-30 days at 26 ℃ under illumination.

(7) Rooting

Selecting differentiated plantlets from the differentiation medium, transferring the plantlets to an MS culture medium containing 1/2 for rooting, culturing the plantlets in light at 28 ℃ for 30 days, and transferring the plantlets to a field for growth.

3. Genetic engineering rice identification

(1) Extraction of genomic DNA

Get T₀Leaves of HPT positive regeneration seedlings are placed into a centrifuge tube about 2cm in length, 600 mu l of CTAB extraction buffer solution (2% CTAB, 1.38MNaCl, 0.1M Tris-HCl, 20mM EDTA, PH8.0) is added, after being crushed by a shaking crusher, the mixture is bathed in a warm bath at 65 ℃ for 60min, chloroform/isoamylol with the same volume is added, the mixture is inverted and mixed gently, and the mixture is centrifuged at 12000rpm for 10 min; transferring the supernatant into another new 1.5ml centrifuge tube, adding equal volume of isopropanol, slowly inverting and mixing, and standing at room temperature for 60 min; 12000rpCentrifuging for 10min, removing supernatant, rinsing DNA precipitate with 70% ethanol, air drying, adding 80 μ l TE buffer solution to dissolve DNA, and storing at-20 deg.C;

(2) PCR amplification

Extracting genome DNA from leaves in a seedling stage, carrying out PCR amplification by adopting a human chymotrypsinogen forward primer Gt13a-F (SEQ ID NO.2: 5'-CACATCCATCATTATCCATCCACC-3') and a reverse primer CT-R (SEQ ID NO.3: 5'-ACTTCGGGTTCTTGAACACCTT-3'), wherein the theoretical size of a product is 547bp, and identifying a gene engineering plant containing a target gene by PCR, wherein the PCR detection method comprises the following steps:

1) taking 1 mul of rice genome DNA as a template, and setting positive (plasmid DNA) and negative (sterile water) controls;

2) the PCR amplification reaction system contained 2.5. mu.l of 10 XBuffer; mu.l of 5U/. mu.l rTaq enzyme, 4. mu.l of 2.5mM dNTP, 0.5. mu.l each of forward and reverse primers; add ddH₂O to 25. mu.l.

3) Pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and final extension at 72 ℃ for 10 min.

4) And (3) carrying out electrophoresis on the PCR amplification product by using 1% agar gel, and observing the result in a gel imager after EB staining.

The identification result shows that 56 independent recombinant human chymotrypsin pro-genetic engineering rice strains are obtained through agrobacterium tumefaciens mediated transformation. The PCR identification result of the target gene of part of the genetically engineered rice is shown in FIG. 4.

4. Identification of expression amount of recombinant human chymotrypsinogen

Transplanting the obtained recombinant human chymotrypsinogen positive seedling into a greenhouse to grow to be mature, and harvesting single plants capable of normally fructifying. As no commercially available detection test kit for quantitatively detecting the content of the recombinant human chymotrypsinogen exists at present, high-expression single plants are preliminarily screened by an SDS-PAGE method, and negative single plants are rejected. And continuously planting the high-expression single plant serving as a candidate single plant, and performing expression quantity comparison and homozygote screening again until a homozygote plant line for stably and efficiently expressing the recombinant human chymotrypsinogen is obtained. The results of SDS-PAGE detection of the expression level of a part of recombinant human chymotrypsinogen rice are shown in FIG. 5. Through preliminary estimation, the maximum expression quantity of the recombinant human chymotrypsin can reach more than 2g/kg brown rice.

SEQ ID NO.1：

SEQ ID NO.2:

5’-CACATCCATCATTATCCATCCACC-3’

SEQ ID NO.3:

5’-ACTTCGGGTTCTTGAACACCTT-3’

[ EXEMENT 2 ] extraction of recombinant human chymotrypsinogen from genetically engineered rice

1. Preliminary screening of recombinant human chymotrypsinogen extraction conditions

Husking genetically engineered rice, processing into semi-polished rice, and grinding into rice flour of 80-100 meshes. Rice flour was mixed with sodium phosphate dibasic-citric acid extraction buffers of different pH at a ratio of 1:5 (weight/volume, kg/L) and extracted according to the extraction conditions as designed in Table 1. After extraction, the supernatant was subjected to SDS-PAGE. The results showed that there was no significant difference in the amount of the target protein extracted from the extract solution for each extraction condition (see FIG. 6). Further analysis shows that the impurity proteins of the

experimental treatments

2, 3, 6, 9, 20 and 26 are relatively less, and the high molecular impurity proteins are almost absent. Wherein the extraction time is 8.5h for treatment 3 (pH of the extract is 3.0, 0mM NaCl), and the purity is relatively high at the extraction temperature of 17 ℃. From the above results, it can be seen that pH has no significant effect on the extraction rate of the target protein, but has a significant effect on the purity of the target protein in the extract, and the lower the pH, the less impurities; the concentration of sodium chloride, the extraction time, the extraction temperature and the like have no obvious influence on the extraction rate of the target protein.

TABLE 1 design of different extraction conditions

2. Determination of extraction conditions of recombinant human chymotrypsinogen

And further optimizing the extraction conditions according to the preliminary screening result. Husking the genetically engineered paddy to prepare semi-polished rice, and grinding the semi-polished rice into rice flour of 80-100 meshes. Mixing rice flour at a ratio of 1:5 (weight/volume, kg/L) with disodium hydrogen phosphate-citric acid extraction buffer solution of pH2.5, pH3.0 and pH3.5 respectively, and extracting at 25 deg.C for 1 hr. Meanwhile, an extraction buffer solution with the pH of 3.0 is designed to be stirred and extracted for 1h, 2h and 4h at the temperature of 20 ℃,25 ℃ and 30 ℃. After extraction, centrifugation was carried out, and the supernatant was subjected to SDS-PAGE. The results showed that there was no significant difference in the amount of the target protein extracted from the extract under each extraction condition (fig. 7). From the economic point of view, the shorter the extraction time, the temperature of 20-30 ℃ (room temperature) and the lowest cost are preferred. Therefore, the extraction conditions of the recombinant human chymotrypsinogen were determined: the pH value of a buffer solution (disodium hydrogen phosphate-citric acid) is 2.5-3.5; the extraction time is 1-2 h; the extraction temperature is 20-30 ℃.

Example 3 Primary purification of recombinant human chymotrypsinogen Using cationic chromatography Medium

1. Selection of primary purification cationic chromatography media

Crude extracts containing recombinant human chymotrypsinogen were prepared according to the extraction conditions determined in example 2 and then loaded onto chromatography columns packed with Unigel80 SP, Unigel80CM, Unigel MMC50S chromatography media, respectively, followed by gradient elution with disodium hydrogen phosphate-citric acid buffers of different pH. The results show that the elution peak of the 3 chromatographic media is relatively seriously trailing and the separation degree is relatively poor when the chromatographic media are eluted by pH gradient. The results of the electrophoresis are shown in FIG. 8, and the penetration of Unigel80CM is evident (FIG. 8B), i.e.the load 20CV is overloaded. In the Unigel MMC50S, tailing is serious, obvious target protein shedding is seen at the tailing part (figure 8C), and target protein is eluted under various pH conditions and is mainly concentrated in elution components with pH6.0 and pH 7.0. No target protein was removed during loading and re-equilibration of Unigel80 SP, which was mainly concentrated in the elution fractions at pH4.5 and pH5.0, but the two conditions gave severe tailing of the elution peaks. Given the low loading of Unigel80CM and the severe re-equilibrium tailing of Unigel MMC50S and the loss of the target protein, Unigel80 SP was ultimately preferred as the chromatography medium for primary purification.

2 cationic Unigel80 SP chromatographic load determination

Crude extracts (i.e., supernatants) of recombinant human chymotrypsinogen were prepared according to the extraction conditions determined in example 2. The crude extract of recombinant human chymotrypsinogen was loaded (0.5ml/min flow rate) onto a 1ml Unigel80 SP column and when loaded to 20CV the breakthrough fluid was automatically collected using a distribution collector at approximately 2 ml/tube. The collected lysates were subjected to SDS-PAGE, and as a result, the target proteins were observed to permeate through 74CV (20+ 27X 2: 74CV) which is 27 tubes of the collected lysates, as shown in FIG. 9. Thus, under this loading condition, the maximum loading of Unigel80 SP was 74 CV.

3 cation Unigel80 SP chromatography optimization

Crude extracts of recombinant human chymotrypsinogen were prepared according to the extraction conditions determined in example 2. 50ml (50CV) of crude recombinant human chymotrypsinogen extract was loaded onto 1ml Unigel80 SP chromatography medium and then eluted with a gradient of 0-200 mM NaCl in disodium hydrogenphosphate-citric acid buffer pH 4.0. As shown in FIG. 10, a trace amount of the target protein eluted under the 0mM NaCl wash at pH4.0, and the target protein was significantly eluted from the chromatographic medium when the salt concentration was increased to 30mM NaCl. From the above, the target protein is very easy to elute, and the washing space is very small. Considering that the target protein is lost more and has tailing phenomenon when the target protein is washed by 30mM sodium chloride at pH4.0 and 0mM, more impurity protein can be removed and the washing peak has a certain separation degree with the subsequent elution peak (pH4.0 and 100mM sodium chloride elution) when the target protein is washed by 0.0 and 0mM sodium chloride, the washing condition is determined to be pH4.0 and 0mM sodium chloride. Since there was no significant difference in the purity of the target protein in the two elution fractions, pH4.0, 100mM sodium chloride elution (collection volume 27CV) and pH4.0, 150mM sodium chloride elution (collection volume 25CV), the elution conditions were determined to be pH4.0, 100 to 150mM sodium chloride, preferably pH4.0, 150mM sodium chloride elution (elution volume is relatively small).

Example 4 Fine purification of recombinant human chymotrypsinogen Using anion chromatography Medium

The primary product of recombinant human chymotrypsinogen was prepared according to the extraction and cation chromatography conditions defined in examples 2 and 3, and since the first primary purification step employs a cationic medium, the second fine purification step is intended to employ an anionic medium.

1. Preliminary product pretreatment

Firstly, properly concentrating a primary product by using a 5-10 kDa ultrafiltration membrane (polyether sulfone material), dialyzing by using a disodium hydrogen phosphate-citric acid buffer solution or a phosphate buffer solution with the pH value of 7.9-8.1 and the electric conductivity of 1.5-2.0 mS/cm until the electric conductivity is 1.5-2.5 mS/cm, and adjusting the pH value to 7.9-8.1 to be used as a sample loading solution for the step 2 chromatography.

2. Anion chromatography medium screening

The pretreated liquid samples obtained in step 1 are respectively loaded into a chromatographic column filled with 1ml of anion chromatographic medium Unigel80Q, NanoGel 50Q, MMA-50S and Q Bestrose FF at the flow rate of 0.5ml/min, and then the chromatographic column is subjected to sodium chloride gradient elution. The result is shown in fig. 11, the binding of the target protein and the Q-series chromatographic medium is weak, the target protein is gradually eluted at a lower conductance (25-50 mM sodium chloride), and the phenomenon of tailing of the elution peak is serious, especially Q best FF, 25mM sodium chloride can elute part of impurities, but the target protein cannot be shed. The results showed that the purity of the protein of interest in the final eluate was essentially the same regardless of the anion chromatography medium (FIG. 11). Unigel80Q is preferred as the step 2 chromatographic medium from the viewpoint of cost and durability of the chromatographic medium.

Unigel80Q chromatography optimization

A chromatographic load according to example 4 was prepared according to the conditions determined in examples 2 to 3, and then applied to a 1ml Unigel80Q column at a loading rate of 40ml (40CV), followed by elution with a disodium hydrogenphosphate-citric acid buffer (or a10 mM phosphate buffer) containing 50mM sodium chloride at pH7.9 to 8.1. As a result, as shown in FIG. 12, the Unigel80Q eluate showed three protein bands in the reduced state, and a single band in the non-reduced state. Through analysis, the recombinant human chymotrypsinogen may have self-activation phenomenon in the process of protein expression, transportation and storage.

Since the collection volume is large when the elution is carried out under the chromatography conditions, which indicates that the elution strength is low, the anion chromatography can improve the elution strength by reducing the elution pH, and therefore, the elution conditions of the chromatography are supposed to be optimized by reducing the pH of the eluent. As shown in Table 2, the collection volume was greatly reduced when the elution was carried out using 50mM sodium chloride, 10mM phosphate buffer, pH 6.5. After optimization of the elution conditions, the purity of the sample did not change significantly (see table 2 and fig. 13).

In conclusion, the Unigel80Q chromatographic elution conditions of the recombinant human chymotrypsin are as follows: 10mM phosphate buffer, pH 6.5-8.0, 25-50 mM sodium chloride. Optimal activation conditions for recombinant human chymotrypsin in conjunction with example 6 were: the concentration of the recombinant human chymotrypsinogen is 1mg/ml, and the pH value is 8.0. The optimal elution conditions for Ungel 80Q were 10mM phosphate buffer, pH8.0, 50mM sodium chloride (the protein content of the collection was about 1mg/ml under these elution conditions, and the collection was used directly for activation without pH adjustment).

TABLE 2 chromatographic elution Collection volumes for different elution conditions

Example 5 verification of three-batch Small-Scale experiments for recombinant human chymotrypsinogen

Three batches of small-scale process verification are carried out according to the optimal extraction, primary purification and fine purification conditions determined in the embodiments 2-4, and the verification process and the verification results are as follows.

1. Preparation (extraction) of crude extract of recombinant human chymotrypsinogen

Weighing 500g of recombinant human chymotrypsinogen rice flour, adding 2500ml of extraction buffer solution containing 14.72g/L disodium hydrogen phosphate dodecahydrate and 16.69g/L citric acid monohydrate, and stirring and extracting at 20-30 ℃ for 1-2 h. After extraction, 2-5% (w/v) of filter aid is added, and filter pressing is carried out by adopting a positive pressure filtration mode. Filtering the primary filtrate with 0.22 μm filter membrane to obtain crude extract of recombinant human chymotrypsinogen (Unigel 80SP chromatography sample liquid).

2. Preliminary purification of recombinant human chymotrypsinogen

20.41g/L disodium hydrogen phosphate dodecahydrate and 15.02g/L citric acid monohydrate buffer solution with the pH value of 3.3-3.5 of 4-6 CV are filled with Unigel80 SP at a linear flow rate of 300-600 cm/h in a balanced mode, and the packed column is an XK16/40 chromatographic column with the column height of 19-21 cm. And (3) taking the crude recombinant human chymotrypsinogen extract as a loading solution for loading, wherein the loading volume is 50 CV. Eluting the impurity protein by using a buffer solution which is 8-10 times of the column volume and has the pH value of 3.9-4.1 and contains 7.61g/L disodium hydrogen phosphate dodecahydrate and 12.91g/L citric acid monohydrate at a linear flow rate of 300-600 cm/h; and (3) eluting the recombinant human chymotrypsinogen by using the disodium hydrogen phosphate-citric acid buffer solution containing 150mM sodium chloride with the pH value of 3.9-4.1 at a linear flow speed of 300-600 cm/h, and collecting the eluate containing the recombinant human chymotrypsinogen to obtain a primary product containing the recombinant human chymotrypsinogen.

3. Fine purification of recombinant human chymotrypsinogen

The method comprises the following steps of (1) filling Unigel80Q into a 10-15 times column volume disodium hydrogen phosphate-citric acid buffer solution with the pH value of 7.9-8.1 and the conductivity of 1.5-2.0 mS/cm at a flow rate of 382-840 cm/h in a balanced manner, and filling a C10/40 chromatographic column with the column height of 27-29 cm; properly concentrating the recombinant human chymotrypsinogen primary product (namely Unigel80 SP chromatography collection liquid) by using a 5-10 kDa ultrafiltration membrane (polyether sulfone material) and dialyzing by using a disodium hydrogen phosphate-citric acid buffer solution (or a10 mM phosphate buffer solution with the same pH value) with the pH value of 7.9-8.1 and the conductance of 1.5-2.0 mS/cm until the conductance is reduced to 1.5-2.5 mS/cm, and adjusting the pH value to 7.9-8.1 to serve as a sample loading liquid for the chromatography; eluting with a buffer solution with pH of 7.9-8.1 and containing 50mM sodium chloride, 20.41g/L disodium hydrogen phosphate dodecahydrate and 15.02g/L citric acid monohydrate at a flow rate of 382-840 cm/h, and collecting an eluate containing the recombinant human chymotrypsinogen to obtain the recombinant human chymotrypsinogen (a refined product) with the purity of more than 95%. The chromatogram is shown in FIG. 14, and the chromatograms of the three batches of small samples are basically consistent.

The purity and yield detection results are shown in table 3, the purity of three batches of small recombinant human chymotrypsinogen is more than 95%, the average yield can reach 1.2g/kg of genetically engineered rice flour, and 3 batches of RSD values are verified to be less than 10%, which shows that the process is good in consistency and controllable in quality.

TABLE 3 three lots of pilot plant purity and yield

Example 6 activation of recombinant human chymotrypsinogen

The purified recombinant human chymotrypsinogen is inactive, and a part of peptide fragment is cut off by trypsin to form the recombinant human chymotrypsin with biological activity. Therefore, the recombinant human chymotrypsin needs to be activated by means of enzymolysis.

1. Preliminary exploration of activation conditions

Dividing the purified sample (1mg/ml) of the recombinant human chymotrypsinogen prepared in the implementation 5 into a plurality of parts according to 10 ml/tube, respectively adding trypsin (the activity is more than or equal to 2500U/mg) with the final concentration of 0.5%, 0.25%, 0.125% and 0.05% (w/w, trypsin/recombinant human chymotrypsinogen), and then respectively standing and activating at 2-8 ℃ and 25 +/-2.5 ℃. Respectively sampling 1ml of the activated sample for 24h, 32h, 48h, 56h and 72h, adding 2-3 mu l of 50% HCl to terminate activation (pH is 3-4), and carrying out activity detection on the activated sample according to a method described in Chinese pharmacopoeia 2020 edition. The results are shown in table 4, after trypsin with different concentrations is added at the temperature of 2-8 ℃ and respectively activated for 24-72 hours, the enzyme activities of the recombinant human chymotrypsin all reach the requirements of 1000U/mg in the 'Chinese pharmacopoeia' 2020 edition, and the trypsin with different concentrations and the enzyme activities at each activation time point have no obvious difference. But the trypsin with the same concentration is activated for 72 hours at the temperature of 25 ℃, and the activity of the recombinant human chymotrypsin is extremely low and is only 200-300U/mg.

TABLE 4 detection results of activity of recombinant human chymotrypsin under different activation conditions

2 activation Condition optimization

In order to optimize the activation conditions of the recombinant human chymotrypsinogen, the concentration (set to be 1-5 mg/ml) of the recombinant human chymotrypsinogen and the pH (7.0-8.5) of the solution during activation are optimized on the basis. When the adding amount of trypsin is 0.1% (w/w), the pH of the solution is 8.0 during activation, the solution is activated for 40 hours at the temperature of 2-8 ℃, and the activity of the recombinant human chymotrypsin is gradually reduced along with the increase of the concentration of the recombinant human chymotrypsin zymogen before activation (Table 5); when the trypsin is added in an amount of 0.1% (w/w) and activated for 40 hours at 2-8 ℃ under different pH values, the activity of the recombinant human chymotrypsin is relatively highest when the pH value of the activation solution is 8.0 (Table 6). SEC-HPLC of the recombinant human chymotrypsin (pro) before and after activation is shown in FIG. 16 and FIG. 17, the purity of the recombinant human chymotrypsin after activation reaches 95%, and is slightly reduced compared with the purity of the recombinant human chymotrypsin zymogen before activation, which is 99%.

The optimal activation conditions were therefore determined to be: the concentration of the recombinant human chymotrypsinogen is 1-5 mg/ml, preferably 1 mg/ml. The adding amount of the trypsin is 0.05 to 0.5 percent, and the preferential adding amount is 0.1 percent; the activation pH is 7.0-8.5, preferably pH 8.0.

TABLE 5 Effect of chymotrypsinogen concentration on post-activation Activity

TABLE 6 Effect of Activity after different pH activation assay results

Example 8 study of recombinant human chymotrypsin preparation

Chymotrypsin is relatively stable in a solid state, but a solution thereof is unstable, and therefore, in order to facilitate storage, it is necessary to freeze-dry recombinant human chymotrypsin. And (3) properly concentrating the recombinant human chymotrypsin activated by the implementation 7 by adopting a 5-10 kDa ultrafiltration membrane (made of polyether sulfone material), carrying out ultrafiltration dialysis by using ultrapure water until the conductance is below 0.5mS/cm, then filtering by using a 0.22 mu m filter membrane, subpackaging the filtrate, and freeze-drying. The lyophilized product is white crystalline powder, can be rapidly dissolved after adding water, and has potency of 1500U/mg. The electrophoresis detection result of the preparation is shown in fig. 18, wherein the commercially available drug is chymotrypsin for injection (lyophilized powder for injection) produced by the first chemical pharmaceutical company ltd; USP reference substance is USP chymotrypsin standard substance.

In a non-reduction state, the electrophoresis behavior of the recombinant human chymotrypsin is consistent with that of a USP reference substance, and a part of degradation fragments exist in the commercially available drugs; in a reduction state, the recombinant human chymotrypsin presents 3 electrophoresis bands, wherein the macromolecular band is suspected to be a part of unactivated recombinant human chymotrypsinogen.

Example 9 production Process of recombinant human chymotrypsin

The production process of recombinant human chymotrypsin established from examples 1-8 is as follows: planting of genetically engineered rice, rice processing (shelling and milling), extraction, preliminary purification by cation chromatography, fine purification by anion chromatography, activation, concentration, dialysis, blending, sterilization and filtration, filling and freeze drying.

SEQUENCE LISTING

<110> Wuhanhe grass element Biotechnology Ltd

<120> a method for preparing recombinant human chymotrypsin from genetically engineered rice expressing human chymotrypsinogen

<130> WH1190-20P150166

<160> 3

<170> PatentIn version 3.5

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Claims

1. A preparation method of genetically engineered rice for efficiently expressing human chymotrypsinogen comprises the following steps:

(1) synthesizing a rice codon optimized human chymotrypsin protogene with a sequence shown as SEQ ID NO. 1;

(2) introducing the human chymotrypsinogen gene obtained in the step (1), a rice specific promoter Gt13a and a signal peptide thereof into a plasmid vector to construct a rice endosperm cell specific expression human chymotrypsinogen expression vector;

(3) transforming the vector obtained in the step (2) into callus regeneration tissues of rice varieties;

(4) culturing the callus regeneration tissue, and obtaining a genetic engineering rice plant expressing human chymotrypsinogen through screening and inducing;

(5) culturing the genetic engineering rice plant expressing the human chymotrypsinogen to obtain the genetic engineering rice expressing the human chymotrypsinogen.

2. The method according to claim 1, wherein the plasmid vector of step (2) is pOsPMP801 having a structure as shown in FIG. 3.

3. A method for preparing recombinant human chymotrypsin by utilizing genetically engineered rice expressing human chymotrypsinogen comprises the following steps:

(1) extracting a crude extract containing recombinant human chymotrypsinogen from rice expressing human chymotrypsinogen genetic engineering;

(2) subjecting the crude extract containing the recombinant human chymotrypsinogen to cation exchange chromatography to obtain a primary product;

(3) carrying out anion exchange chromatography on the primary product to obtain purified recombinant human chymotrypsinogen;

(4) activating the purified recombinant human chymotrypsinogen by trypsin, and freeze-drying to obtain recombinant human chymotrypsin;

the method obtains the recombinant human chymotrypsin with the purity of 95 percent;

wherein the genetically engineered rice grain expressing human chymotrypsinogen is prepared according to the method of claim 1.

4. The method according to claim 3, wherein the crude recombinant human chymotrypsinogen extract of step (1) is prepared by the following steps:

1) husking the recombinant human chymotrypsin pro-genetic engineering paddy rice, processing the husked paddy rice into semi-polished rice, and grinding the semi-polished rice into rice flour of 80-100 meshes;

2) mixing the rice flour and the extraction buffer solution according to the weight/volume ratio of 1:5, wherein the unit is kg/L; extracting for 1-16 hours at 4-30 ℃; the extraction buffer solution comprises the following components: 4.44-69.66 g/L disodium hydrogen phosphate dodecahydrate and 0.58-19.71 g/L citric acid monohydrate, and the pH value is 2.5-8.0;

3) filter-pressing the mixture extracted in the step 2) with 2-5% of perlite, and filtering the filtrate with a 0.22 mu m filter membrane to obtain the crude extract of the recombinant human chymotrypsinogen.

5. The method according to claim 4, wherein the extraction buffer solution of the recombinant human chymotrypsinogen is disodium hydrogen phosphate-citric acid buffer solution with pH of 2.5-3.5, the extraction time is 1-2 h, and the extraction temperature is 20-30 ℃.

6. The method of claim 3, wherein the cation exchange chromatography medium of step (2) is SP Bestrose FF, Unigel80 SP, Unigel80CM, or Unigel MMC 50S.

7. The method of claim 6, wherein the cationic chromatography medium is Unigel80 SP.

8. The method of claim 3, wherein the recombinant human chymotrypsinogen primary product of step (2) is prepared by:

1) balancing a Unigel80 SP chromatographic column at a linear flow rate of 300-600 cm/h by using a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.0-3.5 and the column volume CV of 4-6 times;

2) taking the recombinant human chymotrypsinogen crude extract as a loading solution, wherein the pH of the loading solution is 3.0-3.5, and the loading volume is not more than 75 CV;

3) eluting the impurity protein by using a disodium hydrogen phosphate-citric acid buffer solution containing 0-30 mM sodium chloride and having the pH value of 3.5-4.0 and the CV of 5-10 CV at the flow rate of 300-600 cm/h; the washing buffer solution is pH4.0 and does not contain sodium chloride;

4) eluting the recombinant human chymotrypsinogen by using a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.9-4.1 and containing 100-200 mM sodium chloride at a linear flow rate of 300-600 cm/h, and collecting the eluate rich in the recombinant human chymotrypsinogen to obtain a primary product containing the recombinant human chymotrypsinogen.

9. The method of claim 3, wherein the anion exchange chromatography medium of step (3) is Unigel80Q, NanoGel 50Q, MMA-50S, Q Bestrose FF, Unigel 30Q, or Q Bestrose HP.

10. The method of claim 9, wherein the cationic chromatography medium is Unigel 80Q.

11. The method according to claim 3, wherein the purified recombinant human chymotrypsinogen of step (3) is prepared by:

1) balancing a Unigel80Q chromatographic column at a flow rate of 382-840 cm/h by using 10-15 CV of pH 7.9-8.1 and 1.5-2.0 mS/cm conductance disodium hydrogen phosphate-citric acid buffer solution or phosphate buffer;

2) properly concentrating the primary product by using a 5-10 kDa ultrafiltration membrane (polyether sulfone material), dialyzing by using a disodium hydrogen phosphate-citric acid buffer solution or a phosphate buffer solution with the pH of 7.9-8.1 and the conductance of 1.5-2.0 mS/cm until the conductance of the sample liquid is reduced to 1.5-2.5 mS/cm, adjusting the pH to 7.9-8.1, and loading to anion exchange chromatography;

3) eluting with a disodium hydrogen phosphate-citric acid buffer solution or a phosphate buffer solution with pH of 6.5-8.0 and 25-75 mM sodium chloride at a flow rate of 382-840 cm/h, and collecting an eluate containing the recombinant human chymotrypsinogen to obtain the purified recombinant human chymotrypsinogen with the purity of more than 99%.

12. The method according to claim 3, wherein the recombinant human chymotrypsin of step (4) is prepared by:

1) adding an activating agent trypsin into the purified recombinant human chymotrypsinogen of 0.75-4 mg/ml according to the proportion of 0.05-0.5% (w/w), standing and activating for 36-72 h at the temperature of 2-8 ℃;

2) after the activation is finished, adjusting the pH value to 2.5-3.5, and stopping the activation;

3) properly concentrating the activated recombinant human chymotrypsin by using a 5-10 kDa ultrafiltration membrane made of polyether sulfone, dialyzing by using ultrapure water until the conductance is reduced to be below 0.5mS/cm, and adjusting the pH to be 5.5-6.5; and (5) loading into a tray, and freeze-drying to obtain the recombinant human chymotrypsin freeze-dried powder.

13. A recombinant human chymotrypsin prepared according to the process of any one of claims 3 to 12.